Short communication Valerie A. Pferdeort 1 Thomas K. Wood 2 Kenneth F. Reardon 1, 3 1 Department of Chemical Engineering, Colorado State University, Fort Collins, CO, USA 2 Departments of Chemical Engineering and Molecular & Cell Biology, University of Connecticut, Storrs, CT, USA 3 Department of Environmental & Radiological Health Sciences and Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, CO, USA Proteomic changes in Escherichia coli TG1 after metabolic engineering for enhanced trichloroethene biodegradation Through metabolic engineering, new enzymatic pathways can be introduced into cells to enable or enhance production or biotransformation of chemicals. However, these changes have physiological consequences that can be important but are not well understood. Here we describe the use of two-dimensional gel electrophoresis (2-DE) to detect changes in the proteome of Escherichia coli cells that have been engineered to transform the pollutant trichloroethene (TCE) with the enzyme toluene o-monooxy- genase (TOM). Comparison of 2-DE gels (isoelectric point range 4–7) for E. coli cells with and without the ability to synthesize TOM revealed 31 new proteins in TOM-con- taining cells as well as nine proteins not detected in those cells but present in the plas- mid control strain. Exposure of TOM-containing cells to TCE led to the synthesis of four new proteins and the loss of only one protein. Thus, this example of metabolic engi- neering has a substantial and complex impact on the physiology of these cells that was clearly revealed using a proteomic approach. Keywords: Biodegradation / Escherichia coli / Metabolic engineering / Plasmid / Trichloro- ethene PRO 0418 Trichloroethene (TCE) is a suspected human carcinogen and one of the most frequently reported groundwater pol- lutants [1]. Although this contaminant can be biode- graded anaerobically by reductive dechlorination, the proper conditions for complete dechlorination are often not achievable and the accumulation of lesser-chlori- nated ethenes, including the more toxic vinyl chloride, can occur [2]. TCE can also be degraded aerobically by cometabolism, which involves the nonspecific action of oxygenases. However, aerobic cometabolism of TCE is problematic to achieve because it requires the addition of an inducer for the responsible enzyme (toluene-o- monooxygenase, TOM) and an energy source, and be- cause a cytotoxic intermediate (TCE epoxide) is gener- ated [3, 4]. Metabolic engineering involves genetic manip- ulations to alter enzymatic, regulatory, and/or nutrient transport activities within cells [5]. This approach offers a means to overcome the shortcomings of aerobic cometa- bolism by manipulating the promoter to allow uninduced production of TOM, evolving the activity of TOM to achieve higher rates of TCE degradation, and cloning an additional enzyme to react with and protect the cell from toxic intermediates. However, the production of new enzymes and/or changes in a regulatory mechanism can have unforeseen effects on the physiology of the host organism, with possibly detrimental consequences to the desired application [5, 6]. Unfortunately, few analyses of the effects of metabolic engineering have focused on overall changes in host cell physiology as measured by changes in protein production or other cellular processes. Since most cellular processes are either regulated or directly carried out by proteins or protein complexes, physiological responses to new genes can be expected to result in altered production of various host cell proteins other than those introduced in the genetic manipulation [7]. Thus, proteomic analyses of metabolically engineered cells are expected to yield valu- able insights into the overall cellular pathways that are affected by metabolic manipulation. Specifically, 2-DE offers an ideal approach to study changes in a subpopu- lation of a cell’s proteome because of its ability to resolve complex protein mixtures into individual polypeptides. The first step of this project involved construction of the Escherichia coli TG1 pBS(Kan-)/TOM clone which consti- tutively expresses the genes for the six subunits of the Correspondence: Prof. Kenneth F. Reardon, Department of Chemical Engineering, Colorado State University, Fort Collins, CO 80523-1370, USA E-mail: reardon@engr.colostate.edu Fax: 11-970-491-7369 Abbreviations: Kan, Kanomycin; TCE, trichloroethene; TOM, toluene-o-monooxygenase 1066 Proteomics 2003, 3, 1066–1069  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0606–1066 $17.501.50/0 DOI 10.1002/pmic.200300418